20.106J – Systems Microbiology
Lecture 7
Prof. DeLong
¾ Problem set 2 due today.
¾ For next week: we’ll be doing Chapter 8 of Brock
¾ Exam in a week and a half
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Slide: Central Dogma
o Replication, Transcription, Translation
o Reverse transcription sometimes
Slide: Flow of information
o Right now we’re just thinking about information flow within an individual
species – how nucleic acid information flow occurs
o How you maintain fidelity and copy your DNA for all your daughter cells
What we know about DNA:
o The four bases
o The ring system of the sugar
ƒ The number of carbons
ƒ How you tell the difference between DNA and RNA (the lack of
oxygen)
o The nomenclature of the bases
o Alternating linkage (3’ and 5’)
o The base pairing (G-C, A-T) –This is how it replicates with such high
fidelity.
o Anti-parallel (5’ Æ 3’, 3’ Æ 5’)
o When the structure was first determined, the pairing was inferred, but the
method of replication wasn’t clear
ƒ It could have been Semiconservative, Conservative, or Random
Dispersive.
ƒ These different methods incorporate the mother strands into the
daughter strands in different ways.
ƒ To determine this, they added 15N labels experimentally – they
could predict the densities resulting from each replication method
– heavy vs. light
ƒ The Conclusion, of course, was that it was semiconservative.
o The replication process is not simple, and involves a lot of enzymes.
ƒ Polymerase III can’t just set down on a DNA strand – it needs a
DNA primer. That primer is then used to extend the DNA strand.
ƒ This means that initially it’s not just a DNA strand – it’s a DNARNA hybrid.
ƒ The DNA polymerase synthesizes 5’ Æ 3’. This means that one
strand goes more easily than the other strand. The leading strand
goes directly, but the lagging strand develops a lot of smaller
fragments – these are called Okazaki fragments (about 1000 bases
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long). DNA polymerase I (not III) is used on the lagging strand, so
that it will eat through the extra primers (polymerase I would stop).
ƒ An RNA primer is laid down by a primase.
ƒ The primers have to be removed.
ƒ Nicks have to be removed by DNA ligase.
ƒ With a circular DNA, the replication happens circularly (a bubble
opens up between the strands, and replication happens there).
• This is generally what happens with prokaryotes.
• But there are also some that have linear DNA.
Regulatory Pathways in prokaryotes – the larger topic of today’s lecture.
o The more important means of cell regulation is transcription.
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Prokaryotic transcription
o DNA is more stable than RNA, while RNA can be turned over and used
faster than DNA.
o The 2’ hydroxy group of RNA is useful here.
o RNA polymerase does not need a primer – it just transcribes directly.
o Halfway through the sigma factor falls off.
o Pribnow box and -35 region.
o Some bases are more highly conserved than others. Also, some organisms
are more GC-rich than others.
o There are different types of sigma factors
ƒ Sigma 70 – kind of the vanilla flavor – used for “normal”
promoters
ƒ Sigma 32 is used for heat-shock promoters
ƒ Sigma 54 is used for N limitation promoters
o There’s a modularity in how cells can turn on all kinds of different genes.
o Strong versus weak promoters.
o Initially RNA polymerase binds, the strand is opened up, and transcription
begins.
o Transcription termination
ƒ Inverted repeats – these connect to each other, stopping
transcription, and they require no extra factors.
ƒ Row dependent terminations – require extra proteins
ƒ There are other sorts as well
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Differences between eukaryotic and prokaryotic transcription
o Archaea are very different from bacteria – they do it more like eukaryotes.
o Eukaryotes do not have classical operons
o Eukaryotic mRNAs are usually sliced, capped, and tailed, in the nucleus
o RNA polymerase structure/function differ
o Initiation complexes differ
o These differences make good targets for antibiotics
o Translation – prokaryotes are better in terms of rapid response –
transcription and translation happen next to each other – they are coupled.
In eukaryotes, one is in the nucleus, the other in the cytoplasm.
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Overview of Prokaryotic Translation
o Slide: simple structure of a prokaryotic gene
o Maintaining high enough fidelity for translation is really remarkable
o Shine-Dalgarno sequence
o The genetic code has been inferred from lots of different experiments –
which codons code for which amino acids
ƒ How this code could have occurred is really remarkable
ƒ Originally it was assumed to be random, in the 1960s, but this
doesn’t seem to be the case, because it seems like there is some
order to the groupings.
ƒ Nobody really knows how it developed yet though.
o Transfer RNAs
o The early experiments that discovered how this works were really
remarkable
o The Genetic code is degenerate (meaning that multiple codons can code
for the same amino acid).
ƒ Sometimes there is a bias (in once cell, for example, UUU might
be used for phenylalanine more often than UUC is used)
ƒ Codon families exist.
ƒ Also codon pairs.
o Staying in the correct frame is critical.
o The code is not absolutely hard and fast:
ƒ Recently selenocysteine and pyrrolysine have been dubbed the 21st
and 22nd amino acids
ƒ Previously people had thought that the Selenium in Selenocysteine
was added later, but this is incorrect. It has its own codon (UGA –
normally nonsense/stop codon!) and transfer RNA.
ƒ For pyrrolysine, it looks like a similar mechanism, although it’s not
yet absolutely hard and fast. (UAG codon)
o The tRNA has to be very well matched (very specific covalent bond).
o Aminoacyl-tRNA Synthetase recognizes both the amino acid and the
tRNA.
ƒ It’s a two-step reaction.
ƒ Anticodon loop and acceptor stem of the tRNA are both involved.
o Messenger RNAs are very labile – they turn over on the order of minutes
in the cells, getting eaten up by nucleases. Other RNAs are more stable.
o Drugs that inhibit translation: there are a whole series of antibiotics that
inhibit bacterial translation without touching eukaryotic translation.
o Initiation
ƒ 50S
ƒ 30S
ƒ Shine-Delgarno
o Elongation
ƒ Movement of ribosome along the strand.
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After peptide bond formation, there’s a new empty site, and the
ribosome moves along so that the chain grows.
ƒ Incredible that the ribosome can do this with such high fidelity.
o Ribozymes are catalytic RNAs